The ultimate objective of this work is to understand how hormones and their receptors interact with coactivators and corepressors to regulate transcription of target genes in a tissue- and hormone-specific manner. The aim of this proposal is to understand how prolactin (PRL) augments progesterone-dependent transcription of the uteroglobin (UG) gene by regulating the binding of RUSH-1alpha (Beta) to the UG promoter. RUSH-1alpha (Beta) are newly identified members of the SWI/SNF family, the first nuclear receptor coactivators to be described. This goal will be achieved by 1) mapping the minimal DNA-binding domain of RUSH-1alpha (Beta) proteins and defining their cognate binding site on the UG promoter; 2) determining whether PRL alone or in combination with progesterone regulates phosphorylation of RUSH-1alpha (Beta) proteins; 3) to evaluate the DNA binding properties of RUSH-1alpha(Beta) proteins and determine whether the RUSH proteins are functionally antagonistic. The minimal DNA-binding domain will be mapped by testing the DNA-binding activity of a panel of N- and C-terminal truncation mutants of RUSH-1alpha(Beta) that expressed in vitro. The cognate binding site of RUSH-1alpha(Beta) will be defined by testing a series of 8-overlapping double stranded oligomers that span the length of UG99 (-170/-85), a subfragment of the UG promoter, in gel shift assays with recombinant RUSH-1alpha(Beta) protein. Probes that give positive gel shift results will be used for DNase I footprinting. Temporal activation of RUSH-1alpha and RUSH-1Beta by PRL plus/minus progesterone will be evaluated in nuclear extracts of HRE- H9 cells by gel shift assays with antipeptide antibodies to unique C- terminal regions of the proteins. Confocal immunocytochemistry will provide high resolution analysis of nuclear localization of RUSH proteins. The effects of PRL on tyrosine or serine-threonine- phosphorylation of RUSH-1alpha(Beta) will be evaluated in vitro with specific inhibitors and antiphospho-tyrosine antibodies. Native RUSH proteins will be isolated by phosphocellulose chromatography, heparin- agarose chromatography and a Biotin/Streptavidin affinity system with a biotinylated-DNA fragment that contains multimerized copies of the RUSH-1alpha(Beta) binding site. RUSH-DNA binding will be evaluated by immunoprecipitation, gel shift assays, kinetic studies, and DNA-dependent ATPase assays. Understanding how RUSH, a coregulator of UG gene trancription, is modulated by PRL and progestrone is central to defining the mechanism of steroid hormone action in the preimpnantation uterus, and may be critical to good reproductive health.